Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2% or 2,000 genes, are estimated to code for GPCRs. Of these, there are approximately 100 GPCRs for which the endogenous ligand that binds to the GPCR has been identified. Because of the significant time-lag that exists between the discovery of an endogenous GPCR and its endogenous ligand, it can be presumed that the remaining 1,900 GPCRs will be identified and characterized long before the endogenous ligands for these receptors are identified. Indeed, the rapidity by which the Human Genome Project is sequencing the 100,000 human genes indicates that the remaining human GPCRs will be fully sequenced within the next few years. Nevertheless, and despite the efforts to sequence the human genome, it is still very unclear as to how scientists will be able to rapidly, effectively and efficiently exploit this information to improve and enhance the human condition. The present invention is geared towards this important objective.
Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors. This distinction is not merely semantic, particularly in the case of GPCRs. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed. Thus, the orphan GPCRs are to the pharmaceutical industry what gold was to California in the late 19th century—an opportunity to drive growth, expansion, enhancement and development. A serious drawback exists, however, with orphan receptors relative to the discovery of novel therapeutics. This is because the traditional approach to the discovery and development of pharmaceuticals has required access to both the receptor and its endogenous ligand. Thus, heretofore, orphan GPCRs have presented the art with a tantalizing and undeveloped resource for the discovery of pharmaceuticals.
Under the traditional approach to the discovery of potential therapeutics, it is generally the case that the receptor is first identified. Before drug discovery efforts can be initiated, elaborate, time consuming and expensive procedures are typically put into place in order to identify, isolate and generate the receptor's endogenous ligand—this process can require from between 3 and ten years per receptor, at a cost of about $5 million (U.S.) per receptor. These time and financial resources must be expended before the traditional approach to drug discovery can commence. This is because traditional drug discovery techniques rely upon so-called “competitive binding assays” whereby putative therapeutic agents are “screened” against the receptor in an effort to discover compounds that either block the endogenous ligand from binding to the receptor (“antagonists”), or enhance or mimic the effects of the ligand binding to the receptor (“agonists”). The overall objective is to identify compounds that prevent cellular activation when the ligand binds to the receptor (the antagonists), or that enhance or increase cellular activity that would otherwise occur if the ligand was properly binding with the receptor (the agonists). Because the endogenous ligands for orphan GPCRs are by definition not identified, the ability to discover novel and unique therapeutics to these receptors using traditional drug discovery techniques is not possible. The present invention, as will be set forth in greater detail below, overcomes these and other severe limitations created by such traditional drug discovery techniques.
GPCRs share a common structural motif All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell. The general structure of G protein-coupled receptors is depicted in FIG. 1.
Generally, when an endogenous ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular “G-protein.” Although other G proteins exist, currently, Gq, Gs, Gi, and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein. A principal focus of this invention is directed to the transmembrane-6 (TM6) region and the intracellular-3 (IC3) region of the GPCR.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. As shown schematically in FIG. 2, a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”
For instance, it was shown that interchange of a short homologous sequence of amino acids in the carboxy terminal of the third cytoplasmic loop of the Gq-phospholipase G protein coupled α1B adrenoreceptor and the Gs-adenyl cyclase-coupled β2 andrenergic receptor resulted in both receptors promoting intracellular activation levels comparable to the fully agonist-stimulated native receptors.
As noted above, the use of an orphan receptor for screening purposes has not been possible. This is because the traditional “dogma” regarding screening of compounds mandates that the ligand for the receptor be known. By definition, then, this approach has no applicability with respect to orphan receptors. Thus, by adhering to this dogmatic approach to the discovery of therapeutics, the art, in essence, has taught and has been taught to forsake the use of orphan receptors unless and until the endogenous ligand for the receptor is discovered. Given that there are an estimated 2,000 G protein coupled receptors, the majority of which are orphan receptors, such dogma castigates a creative, unique and distinct approach to the discovery of therapeutics.
Information regarding the nucleic acid and/or amino acid sequences of a variety of GPCRs is summarized below in Table A. Because an important focus of the invention disclosed herein is directed towards orphan GPCRs, many of the below-cited references are related to orphan GPCRs. However, this list is not intended to imply, nor is this list to be construed, legally or otherwise, that the invention disclosed herein is only applicable to orphan GPCRs or the specific GPCRs listed below. Additionally, certain receptors that have been isolated are not the subject of publications per se; for example, reference is made to a G Protein-Coupled Receptor database on the “world-wide web” (neither the named inventors nor the assignee have any affiliation with this site) that lists GPCRs. Other GPCRs are the subject of patent applications owned by the present assignee and these are not listed below (including GPR3, GPR6 and GPR12; see U.S. Provisional No. 60/094,879):
TABLE AReceptor NamePublication ReferenceGPR123 Genomics 609 (1994)GPR414 DNA and Cell Biology 25 (1995)GPR514 DNA and Cell Biology 25 (1995)GPR728 Genomics 84 (1995)GPR828 Genomics 84 (1995)GPR9184 J. Exp. Med. 963 (1996)GPR1029 Genomics 335 (1995)GPR1532 Genomics 462 (1996)GPR1770 J Neurochem. 1357 (1998)GPR1842 Genomics 462 (1997)GPR20187 Gene 75 (1997)GPR21187 Gene 75 (1997)GPR22187 Gene 75 (1997)GPR24398 FEBS Lett. 253 (1996)GPR3045 Genomics 607 (1997)GPR3142 Genomics 519 (1997)GPR3250 Genomics 281 (1997)GPR40239 Biochem. Biophys.Res. Commun. 543 (1997)GPR41239 Biochem. Biophys.Res. Commun. 543 (1997)GPR43239 Biochem. Biophys.Res. Commun. 543 (1997)APJ136 Gene 355 (1993)BLR122 Eur. J. Immunol. 2759 (1992)CEPR231 Biochem. Biophys.Res. Commun. 651 (1997)EBI123 Genomics 643 (1994)EBI267 J. Virol. 2209 (1993)ETBR-LP2424 FEBS Lett. 193 (1998)GPCR-CNS54 Brain Res. Mol. Brain Res. 152(1998);45 Genomics 68 (1997)GPR-NGA394 FEBS Lett. 325 (1996)H9386 FEBS Lett 219 (1996)HBA9541261 Biochim. Biophys. Acta 121(1995)HG38247 Biochem. Biophys.Res. Commun. 266 (1998)HM745 Int. Immunol. 1239 (1993)OGR135 Genomics 397 (1996)V28163 Gene 295 (1995)As will be set forth and disclosed in greater detail below, utilization of a mutational cassette to modify the endogenous sequence of a human GPCR leads to a constitutively activated version of the human GPCR. These non-endogenous, constitutively activated versions of human GPCRs can be utilized, inter alia, for the screening of candidate compounds to directly identify compounds of, e.g., therapeutic relevance.